This proposal is designed to explore mechanisms operative in control of nucleocytoplasmic RNA transport. Its major thrusts encompass 1) characterization of intron compartmentation in selection of RNA transcripts for transport. Metabolism of introns will be examined using cDNA synthesized with specific polynucleotide promers and subsequently amplified by the polymerase chain reaction. Alterations in splice junctions will be examined following A) treatment with chemical carcinogens, which alter nucleocytoplasmic compartmentation and nuclear structure; and B) in transfection experiments with a hamster cell line which produces functional human immunodeficiency virus Rev protein, which modulates host cell nucleocytoplasmic transport mechanisms via direct interaction with the nuclear scaffold (NS) nucleoside triphosphatase (NTPase). 2) Definition of the role of the 46kD NTPase in nucleocytoplasmic RNA transport. This enzyme is produced from lamins A/C, the major structural proteins of the NS, by cleavage at a conserved tyrosine residue (aa376) near the end of coil-2. Substantial ATP-binding by cloned lamin C preparations has been documented, which shows cooperative (K D =3x10-6 M) and non-cooperative (K D =3x10-6 M) and non-cooperative (K D =2x10-5 M) sites which involve histidine residues. However, cloned lamin C preparations do not show NTPase activity, suggesting that post-translational modifications or other factors are important. Therefore various expression systems and in vitro modifications will be examined for relevance in production of active NTPase. Since NS NTPase activity and the 46 KD lamin fragment are increased 300% following CCl 4 or thioacetamide treatment, without increase in lamin mRNA, and since the 46 kD N-terminus lacks the nuclear localization signal which dictates intranuclear import of lamins, we hypothesize that newly discovered NS proteases are involved in post- translational production of NTPase. These proteases show a marked selectivity for lamins A/C, are stringently regulated by Ca2+, and appear to control NTPase production during mitotic regeneration and following carcinogen treatment. A number of monoclonal antibodies directed against the 46 kDprotein have been developed. These antibodies modulate NS NTPase activity and have shown parallel modulation of RNA transport in vitro. Anti-NTPase antibodies will be used in vitro, and inoocyte microinjection studies to determine the role of the 46 kD NTPase in RNA transport, and the properties of the NTPase will be directly explored. Properties of the NS proteases will be defined, and used to explore their role in production of the NTPase from lamins A/C, using both cloned lamins and NS preparations.